Background: MyoVs are molecular motors widely distributed in eukaryotic cells responsible for membrane trafficking and intracellular transport. Results: The cargo-binding domain from human MyoV paralogs was structurally and biophysically characterized. Conclusion: We identified singular structural changes and molecular events conferring functional differentiation and modulating cargo binding. Significance: This work provides structural insights into cargo recognition and regulatory mechanisms in MyoVs.

Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.

Plasma cholesteryl ester transfer protein (CETP) activity diminishes HDL-cholesterol levels and thus may increase atherosclerosis risk. Experimental evidence suggests CETP may also exhibit anti-inflammatory properties, but local tissue-specific functions of CETP have not yet been clarified. Since oxidative stress and inflammation are major features of atherogenesis, we investigated whether CETP modulates macrophage oxidant production, inflammatory and metabolic profiles. Comparing macrophages from CETP-expressing transgenic mice and non-expressing littermates, we observed that CETP expression reduced mitochondrial superoxide anion production and H2O2 release, increased maximal mitochondrial respiration rates, and induced elongation of the mitochondrial network and expression of fusion-related genes (mitofusin-2 and OPA1). The expression of pro-inflammatory genes and phagocytic activity were diminished in CETP-expressing macrophages. In addition, CETP-expressing macrophages had less unesterified cholesterol under basal conditions and after exposure to oxidized LDL, as well as increased HDL-mediated cholesterol efflux. CETP knockdown in human THP1 cells increased unesterified cholesterol and abolished the effects on mitofusin-2 and TNFα. In summary, the expression of CETP in macrophages modulates mitochondrial structure and function to promote an intracellular antioxidant state and oxidative metabolism, attenuation of pro-inflammatory gene expression, reduced cholesterol accumulation, and phagocytosis. These localized functions of CETP may be relevant for the prevention of atherosclerosis and other inflammatory diseases.

Several Gram-negative bacterial pathogens have developed type III secretion systems (T3SSs) to deliver virulence proteins directly into eukaryotic cells in a process essential for many diseases. The type III secretion processes require customized chaperones with high specificity for binding partners, thus providing the secretion to occur. Due to the very low sequence similarities among secretion chaperones, annotation and discrimination of a great majority of them is extremely difficult and a task with low scores even if genes are encountered that codify for small (<20 kDa) proteins with low pI and a tendency to dimerise. Concerning about this, herein, we present structural features on two hypothetical T3SSs chaperones belonging to plant pathogen Xanthomonas axonopodis pv. citri and suggest how low resolution models based on Small Angle X-ray Scattering patterns can provide new structural insights that could be very helpful in their analysis and posterior classification.